Ionotropic glutamate receptors (GluRs) are molecular pores which facilitate the passage of ions across cell membranes and mediate excitatory signal transmission in the mammalian nervous system. Because of their essential role in normal brain function and increasing evidence that dysfunction of GluR activity mediates CNS diseases and damage during stroke a substantial effort has been directed towards analysis of GluR properties. The AMPA, kainate and NMDA subtypes of ionotropic glutamate receptors are encoded by at least 7 gene families. One area that remains challenging is identification of the subunit composition of native GluRs. The overlapping expression of mRNAs encoding kainate receptor subunits in a brain region specific pattern makes it extremely likely that, similar to AMPA receptors, native kainate receptors exist as heteromeric assemblies most likely with distinct functional properties. Although recombinant kainate receptors from the GluR5-7 gene family have been studied extensively in their homomeric forms, there have been no tests to determine whether these subunits can coassemble with each other. We used two GluR5 selective agonists, the tert-butyl derivative of AMPA (ATPA) and (S)-5-iodowillardiine (I-will) to test for coassembly of GluR5 with GluR6 and GluR7. Our experiments relied on the reduction in polyamine block which occurs for heteromeric receptors containing both pore region edited and unedited Q/R site subunits. Birectifying ATPA and I-will responses for homomeric GluR5(Q) resulting from polyamine block became outward rectifying when GluR6(R) was coexpressed with GluR5(Q), even though GluR6 was not activated by ATPA or I-will, indicating the formation of heteromeric receptors containing both GluR6 and GluR6 subunits. Because I-will also does not activate homomeric kainate receptors assembled from GluR7 we were able to use the same strategy to test for coassembly of GluR5 with GluR7. Since GluR7 does not undergo Q/R site RNA editing it was necessary to first generate the required construct by site directed mutagenesis. When GluR5(Q) was coexpressed with GluR7(R) responses to I-will again showed reduced rectification compared to those for homomeric GluR5(Q) indicating that GluR5 and GluR7 can also coassemble to form heteromeric receptors. To test for coassembly of GluR6 with GluR7 a different approach was required since no subtype selective drugs are available for these subunits. The low affinity of GluR7(Q) for glutamate and kainate; rapid and complete desensitization to these agonists; and the lack of effect of concanavalin A on desensitization for GluR7(Q) made it possible to test for coassembly of GluR7 with GluR6. By recording responses to concentrations of kainate less than 1 mM, which activate GluR6 but are ineffective in activating GluR7, we were again able to show attenuation of rectification indicating formation of heteromers between GluR6(Q) and GluR7(R). Heteromeric kainate receptors containing both GluR5 and GluR6 subunits exhibit novel functional properties including reduced desensitization and faster recovery from desensitization than recorded for homomeric GluR5. Coexpression of GluR6 with GluR5 also enhanced the magnitude of responses to GluR5 selective agonists. In contrast, coassembly of GluR7 with GluR6 markedly decreased the amplitude of agonist responses. An initially unexpected result of our experiments was profound effects on coassembly of GluR5, GluR6 and GluR7 on the amplitude of responses to GluR5 selective agonists. In part this is likely to stem from the requirement for occupancy of multiple subunits by agonist to activate a high probability of receptor gating. Thus in heteromeric receptors containing GluR5 and GluR6 subunits, GluR5 selective agonists will by default achieve lower occupancy levels than non selective agonists. However, we observed upregulation of responses to GluR5 selective agonists on coexpression with GluR6. It is possible that GluR6 acts as a chaperone and when combined with GluR5 the assembly or cell surface expression of GluR5 is facilitated compared to for homomeric receptors. It is also possible that the upregulation of the amplitude of I-will responses is due to an effect of GluR6 on the gating of GluR5. Evidence consistent with such an effect is the observation that coassembly of GluR5 with GluR6reduces the extent of and speeds recovery from desensitization for GluR5 selective agonists. Clearly, much remains to be learned about kainate receptors at many levels, from basic properties to their role in synaptic circuitry and behavior. Our experiments on coassembly of kainate subunits relied heavily on the use of polyamine block as an assay of coassembly of pore region edited and unedited subunits. In a separate series of experiments we have performed an extensive quantitative analysis of polyamine block in GluR6 mutants with amino acid substitutions at two key sites within the pore region: the Q/R site (position 590) and a conserved negative charge in AMPA and kainate receptors located 4 amino acid residues towards the C- terminus (position 594). Surprisingly, introduction of negative charge at the Q/R site increased the Kd for spermine from 1.3 to 4.0 microM (Q590E); the smaller side chains Q590D and Q590N had Kds of 47 and 20 microM. Reductions in spermine affinity were also obtained for the small hydrophobic residues Q590V and Q590A with Kd(0)s of 3.6 and 8.8 microM. Positively charged side chains produced outward rectifying responses similar to those recorded for GluR6(Q) with polyamine free conditions suggesting a complete absence of voltage- dependent block by spermine. Substitution of tryptophan at the Q/R site produced high affinity block, Kd 190 pM; a much smaller increase in affinity was observed for the other aromatic side chains Q590F and Q590Y which had Kds of 0.28 and 0.83 microM. The Q590H mutant gave weakly birectifying responses strikingly different from those for other mutants. When ionization of the His group was increased by raising the external hydrogen ion concentration, responses became outward rectifying. We believe that protons act as permeable blockers for Q590H and that occupancy of the imidazole ring varies with pH and membrane electric field. Neutralization of charge or aromatic residues introduced at the +4 site produced the largest reductions of spermine affinity, with Kds for E594N, E594Q and E594W of 109, 1020 and 2150 microM. In the absence of polyamines, E594K and E594R produced strongly inward rectifying responses while E594Q, E594A and E594W were birectifying. A 2 barrier 1 site model for permeant block allowed quantitative comparisons between mutants. Despite large changes in well depth and barrier heights there was little change in the voltage dependence of block for both Q/R at +4 site mutants. We propose a model with a distributed binding site for polyamines in which the +4 site is located near the entrance to the channel. Of interest such a structure would be similar to that for the KcsA potassium channel for which a near atomic level structure was recently obtained by macromolecular crystallography. We propose that because the location of the +4 site is very shallow compared to the membrane electric field a substantial fraction of the voltage dependence of polyamine block is indirect and results from coupling to movement of permeant ions. A major challenge for the future will be to determine the affinity and molecular identity of the binding site(s) for permeant monovalent cations in AMPA and kainate subtype GluRs.